Bugs, Guts and SpADr David Ong Eng Hui

Consultant GastroenterologistREANIMATE Lead

National University Hospital

Disclosures

• Advisory Boards/educational grants/honoraria:

– Abbvie

– Takeda

– Ferring

– Vifor

– Janssen

REANIMATE Program

“To give new life to”

NUHS Faecal MicrobiotaTransplantation Program

November 2013

REANIMATE Team

• Acknowledgements:

• Nick Chew, Reuben Wong, Calvin Koh, Juanda Leo, Roland Jureen, Jonathan Lee, Alex Soh

• Other hospitals participating:

• CGH, TTSH, SGH

Microbiome• Nobel Laureate Joshua Lederberg

• Collection of microorganisms that cohabit our bodies

• Viruses, fungi, parasites and bacteria

• RNA transcripts outnumber mammalian transcripts 100 to 1

Lederberg J, The Scientist, 2001

Gut Microbiota and Factors

Affecting its Composition

Simren et al. Intestinal Microflora in FBD: A Rome foundation Report. Gut 2014

The Microbial Balance

Hollister et al. Compositional and functional features of GI Microbiome and effects of health. Gastro 2014

Our Microflora Evolves

An example of how bugs can cause problems in the Gut: C. Difficile Colitis

Brody TJ, Khoruts A.

FMT and emerging

applications.

Nat Rev Gastroenterol 2012.

Epidemiology in Singapore

• Paucity of data from Singapore:

– Lim et al reported 2001 to 2006

• incidence of CDI increased from 1.49 to 6.64 per 10,000 patient days in a secondary hospital

– Hsu et al subsequently 2006 to 2008

• incidence of CDI had fallen from 5.56 to 2.99 per 10,000 patient days in public hospitals

Lim, Poh Lian. “Increasing Incidence of Clostridium Difficile-associated Disease, Singapore.” Emerging nfectious Diseases 14, no. 9 (September 2008): 1487–1489.

Hsu, Li-Yang, Thean Tan, Tse Koh, Andrea L Kwa, Prabha Krishnan, Nancy W Tee, and Roland Jureen. “Decline in Clostridium Difficile-associated Disease Rates in Singapore Public Hospitals, 2006 to 2008.” BMC Research Notes 4, no. 1 (2011): 77.

Case 1: Refractory/ relapsing CDI

80 yo lady

CVA, Bedbound

Recurrent admissions for gram negative septicaemia secondary to recto-urethral fistula

Diarrhoea 10-15 times per day; ATN requiring iv fluid and electrolyte replacement; severe excoriations

CD toxin positive with leukocytosis

C.Difficile Relapse

Occurs in 20-60% of successfully treated patients

• Relapse rate similar for vancomycin & metronidazole ~25-30%

• After 1st relapse, subsequent relapse risk ~40%

• 2 or more relapses, risk rises to ~60%

Onset is 1 week - 3 months after treatment

Refractory CDI seen in 10-20%

Huebner ES, Surawicz CM. Treatment of recurrent clostridium difficile diarrhea. GastroenterolHepatol 2006;2:203-8.

Treatment of Multiple Recurrences

• Frustrating, demoralising to caregivers and patient:– Fidaxomicin

– Infusion of monoclonal antibodies active versus CdA and CdB toxins

– Vancomycin - tapering, pulse dosing, sequencing with rifaximin*

– Probiotics - Saccharomyces boulardii, Lactobacillus - unproven#

– Passive treatment with immunoglobulin - anecdotally effective

– Toxin-binding with cholesytramine or colestipol - unproven^

– Fecal reconstitution using donor feces– effective

– Subtotal colectomy … reports of C diff ileitis

*Interruption of recurrent CDAD episodes by serial therapy with vancomycin and rifaximin. Clin Infect Dis 2007;44:846.

#Focus on probiotics for CDAD: focus on Lactobacillus rhamnosus GG and Saccharomyces boulardii. Pharmacotherapy 2007;41(7):1212.

^Treatment of antibiotic-associated pseudomembranous colitis with cholestyramine resin. Johns Hopkins Med J. 1978;143(3):67.@ Efficacy of fidaxomicin versus vancomycin as therapy for Clostridium difficile Infection in individuals taking concomitant antibiotics for

other concurrent infections. Clin Infect Dis. 2011; 53(5):440

Faecal Microbiota Transplantation (FMT)The new kid on the block?

Animals Ancient Medicine

China“Yellow-Dragon Soup” Tong-Jin Dynasty 4th CenturyMing Dynasty 16th CenturyBedoiuns of North Africa

Modern MedicineEISEMAN B, SILEN W, BASCOM GS, KAUVAR AJ. Fecal enema as an adjunct in the treatment of pseudomembranous enterocolitis. Surgery. 1958 Nov;44(5):854-9. PubMed PMID: 13592638.

Coprophagia

Veterinary Medicine: Transfaunication

Results – 81% cured after first infusion and 93.8% after second infusion

13/16 DFI recipients cured after 1st infusion and 15/16 after 2nd

4/13 vanco monotherapy cured

3/13 vanco + PEG lavage cured

Overall cure rate ratio• DFI to vanco alone

– 3.05 (99.9% confidence interval 1.08-290.05)

• DFI to vanco + PEG– 4.05 (99.9% CI 1.21-290.12)

18 who relapse after vanco alone or vanco/PEG received off protocol DFI – 15 cured (11 with one DFI, 4 with

two DFI)

Faecal Microbiota – Improved fecal diversity after FMT

Faecal bacterial diversity assayed using 16s r RNA gene PCR amplification before and after fecal infusion

Improved fecal bacterial diversity 14 days following infusion• Increased Bacteroides (x 2-4),

Clostridia (x 2-4) and Proteobacteria (x~100 ) absolute numbers after DFI (and species diversity within those genera)

n=15 n=9 n=8

First FMT in Singapore - May 2014

Indications for faecal microbiotatransplantation

• C. difficile infection defined as diarrhea (≥3 loose or watery stools per day for at least 2 consecutive days or ≥8 loose stools in 48 hours) and a positive stool test for C. difficile toxin

• Faecal microbiota transplantation can be considered in patients who have had relapsing C difficile infection (≥ three episodes or two relapses of C. Difficile infection requiring hospitalisation) after adequate courses of antibiotic therapy, including:

• >14 days of metronidazole or > 14 days of vancomycin

• FMT may also be considered in patients with refractory CDI, whereby there is inadequate clinical improvement after treatment with first and second-line antibiotics (metronidazole and vancomycin as outlined above)

Contraindications to faecal microbiota transplantation

• Recent chemotherapy

• Human immunodeficiency virus (HIV) infection with a CD4 count of less than 240

• Immunosuppression, such as the prolonged use of prednisolone at a dose of at least 60 mg per day

• Pregnancy

• Use of antibiotics other than for treatment of C. difficile infection at baseline

• Need for vasopressor medications for maintanence of normal blood pressure.

• Admission to an Intensive Care Unit.

Donor Biologic Screening

Blood Tests

Cytomegalovirus (IgG and IgM)Epstein-Barr Virus (VCA IgM, VCA IgG, VCA, antiEBNA)Hepatitis A (total antibodies, and if positive also Hepatitis A IgM)Hepatitis B (HBsAg, antiHBs)Hepatitis C (anti HCV)HIV-1 and HIV-2 (Combined HIV Antigen/Antibody test)Human T-lymphotrophic virus types I and II (HTLV) (antibodies)Treponema pallidum (EIA total antibody)Entamoeba histolytica antibodiesStrongyloides stercoralis antibodies

Faecal Tests

Bacteriological and parasitological evaluation by local standards:Stool cultureOva, cysts and parasites x3Isospora x3 (acid fast)MRSA stoolVRE screeningCP-CRE screeningGiardia antigenClostridium difficile toxinRotavirusCryptosporidium antigenEntamoeba Histolytica antigen

Faecal Processing protocol

Processing

• Approximately 50-60 g faecal material is homogenized in a commercial blender with sterile normal saline solution

• The slurry is passed through a kitchen type stainless steel strainer to remove larger particulate matter

• The resulting material is centrifuged with sterile saline solution

• Sterile pharmaceutical grade glycerol is added to a final concentration of approx. 10%

Storage

• The faecal solution is stored at -80 C

• Before use the solution is thawed on ice for up to 4 h, and sterile saline is added to a final volume of 250 ml

Quality Control

• One drop of the final thawed suspension is cultured anaerobically , expected to yield heavy mixed growth.

Clear, Colourless and Odourlessliquid

Whose Poop to Use?

• Related Donor– Less “icky” factor

– “I’m getting my wife’s stool… part of her now lives in me!”

– Dysbiosis runs in families and a shared environment

• Unrelated Donor– 90-92% vs 70% success

rate in CDI eradication; 9% vs 30% recurrence (Hamilton et al)

• Fresh is Better?– 92% Fresh vs. 90% Frozen

success rate (Hamilton et al)

– Frozen inoculum via NGT 90% cure rate (Youngster et al)

Hamilton MJ et al. Standardized frozen preparation FMT for treatment of C Difficile. Amer Journ Gastroenterol 2012Youngster I et al. . Fecal microbiota transplant for relapsing Clostridium difficile infection using a frozen inoculum from unrelated donors: a randomized, open-label, controlled pilot study. Clin Inf Dis 2014 Jun;58(11):1515-22.

“Top-down or Bottom-up?”The Best Route for Stool Delivery

Youngster I et al. . Fecal microbiota transplant for relapsing Clostridium difficile infection using a frozen inoculum from unrelated donors: a randomized, open-label, controlled pilot study. Clin Inf Dis 2014 Jun;58(11):1515-22.

Lower GIT route found to be more effective than UGIT route in CDI(>85% vs 76%)

Case 2: FMT in an Immunocompromised Host

60 yo male

Myasthenia gravis, ESRD on HD

Myasthenia crisis; complicated by ventilator associated pneumonia and left basilic and subclavianthrombosis

Refractory clostridium difficile infection; WCC 25; diarrhoea 20x/day

Meds: mycophenalate, prednisolone, inotropic support, IVIG

FMT for clostridium difficile infection is safe in immunocompromised patients

• 75 adult and 5 paediatric patients

• Refractory, recurrent and mixed

• HIV, SOT, IBD on immune-modulators

• Overall cure rate 89%

• SAE in 12 patients (15%)– 2 deaths; one related to sedation during FMT

– One mucosal tear

– 6 unrelated infections or short lived diarrheal illness

– 3 flare of IBD

Kelly Cr et al. Fecal microbiota transplant for treatment of Clostridium difficile infection in immunocompromised patients.Am J Gastroenterol. 2014 Jul;109(7):1065-71. doi: 10.1038/ajg.2014.133. Epub 2014 Jun 3.Neemann et al. Fecal microbiota transplantation for fulminant Clostridium difficile infection in an allogeneic stem cell transplant patient.Transpl Infect Dis. 2012 Dec;14(6):E161-5. doi: 10.1111/tid.12017. Epub 2012 Nov 2.

• An IND application is not currently needed for the treatment of refractory C. difficile, provided that the treating physician obtains adequate informed consent from the patient or his or her legally authorized representative for the use of FMT products

• Informed consent should include at a minimum, a statement that the use of FMT products to treat C. difficile is investigational and a discussion of its potential risks

September 2014

The Future of FMT

The Future of FMT

The Future of FMT

Beyond C Difficile…

FMT in IBS and IBD

• Irritable Bowel Syndrome

1. Case reports/series

2. Mainly in IBS-D, some IBS-C

3. Reports improvement in BM and reduced abdominal pain

4. Awaiting results of ongoing 2 RCTs

• Inflammatory Bowel Disease

1. Case series

2. Positive outcomes in Ulcerative Colitis

3. Disappointing for use in Crohn’s Disease

4. Recent data at DDW 2014 from ongoing RCTs were conflicting

Is there a role for FMT in SpA?

Therapeutic Potential of FMT

Smits LP. Therapeutic potential of FMT. Gastro 2013

Microbiota and diet/environment

• High intake of fruit and veg a/w with increased diversity

• Marked differences in fecal microbiota between people brought up in African rural villages from those living in Europe– Greater diversity with life in rural environment

– Predominance of Proteovella vs Bacteroides

• Westernised diet (high in saturated fat and sugar– decreases bacterial diversity esp Firmicutes

Ursell et al, Nutr Rev 2012De Filipo et al, Proct Natl Acad Sci USA, 2010Albenberg and Wu,Gastroenterology, 2014Hold et al, World Journal Of Gastro, 2014

Microbiota and diet/environment

• High protein, low carb/fibre diet– Decrease butyrate in fecal short chain FA concentrations

within 4 weeks– Reduction of Firmicutes– ?long term adherence may lead to increased risk of

colonic disease

• Short term dietary changes tend to produce modest, less permanent changes

• If severe (35% calorie reduction for 6 weeks) increases bacterial diversity

• Increasing bacterial diversity shows reduction in serum sensitive CRP

Russell et al, Am J of Clin Nutr, 2011Cotillard et al Nature 2013

Colorectal Cancer (CRC) and polyposis

Microbiota plays major role in pathogenesis of CRC

High cancer development in colon and rare in SB

Similarity of microbiota in CRC and Crohn’s disease

Reduced diversity in CRC

– Higher Proteobacteria and lower Bacteroidetes

Study of mucosa associated microbiota may be

better than just studying fecal microbiota

Well established association between Strep

gallolyticus

– Found in 20-50% of CRC compared with less than 5% of

normal colons

Rhodes et al. Colorectal Cancer, 2014

Dulal et al. Cancer J, 2014

Colorectal Cancer (CRC) and polyposis

Fusobacterium nucleatum can interact with

E-Cadherin and TLRs, esp when dysplasia

has occurred and covering mucus layer is

lost

Inflammatory effects on cellular signalling

Inhibition of apoptosis

Allen Vercoe et al. Immunol Lett, 2014

Ahn et al. J Natl Cancer Inst, 2013

McCoy et al. PLoS One, 2013

Does the microbiome play a role in SpA?

• Microbiome plays major role in educating immune system

• Strongly implicated in IBD

– Clinical and genetic overlap between IBD and SpA

– Bowel and joint disease in HLA B27/human beta2 microglobulin transgenic rats

• Mechanism by which HLA B27 influences microbiome still unknown

James T Rosenbaum et al, Clin Rheumatol2014

Changes in microbiome linked to IBD

Microbial composition Decrease in alpha diversity

Decrease in Bacteroides and

Firmicute

Increase in

Gammaproteobacteria

Presence of E Coli

Presence of Fusobacterium

Decrease in Clostridia,

Ruminococcaceae,

Bifidobacterium, Lactobacillus

Decrease in F prausnitzii

Microbial Function Decrease in SCFAs, Butyrate

Decrease in butanoate and

propanoatemetabolism

Decrease in amino acid

biosynthesis

Increase in auxotrophy

Increase in Amino acid

tyransport

Increase in oxidative stress

Increase in type II secretion

system, secretion of toxins

Kostic et al, Gastroenterology, 2014

Search of IBD and microbiome yielded more than 900 references

Mouse models

Absence of NLRP6 results in colitis and alteration of intestinal microbiota

Elinav et al, Cell, 2011

NLRP6 knockout mouse

Germ free wild type mouse

Wild type mouse develops inflammation

Co-housed

Where is the evidence that says SpA is microbiome driven?

• Associations between IBD and AS

– 10-20% of patients with IBD develop sacroiliitisidentical to AS

– >50% of patients with AS have microscopic colonic lesions that look like CD

– Periphearl arthritis and uveitis are characteristic of both AS and IBD

Bluestone et al, Ann Rheum Dis 1975Mielans et al, J Rheumatol, 1985

When in doubt look at our genes….

• Large number of overlap genes between IBD and AS

• High proportion involved in mucosal immunity– RUNX3, EOMES, TBX21

– Cytokine receptors IL7R and IL23R

• Key regulators of differentiation and activation of innate lymphoid cells

• Critical components of mucosal immune defenses

Cortes A et al, Nat Gen 2013

Strongest Evidence that microbiome is important in SpA comes from Animal data

Transgenic 33-3 rats

Raised in germ free environment

Colitis and arthritis eliminatedOverexpress HLA B27 and Human beta 2 microglobulin

Develop spontaneous diarrhoea and arthritis

Hammer et al, Cell, 1990Taurog et al, J Exp Med 1994

Probiotics can maintain the remission whilst colonisation with other bacteria can induce

disease

Colitis and arthritis eliminated by germ free environment Probiotics Lactobacillus Rhamnosus GG

Maintain remission

Bacteroides thetaiotamicronBacteroides vulgatus

Induce diarrhoea and arthritis

Dieleman et al, Gut , 2003

Another example of a mouse model….

SKG mouse model

Features of both SpA and IBD

Shows marked involvement of IL23 dependent immunological pathways

Germ free environment

Unaffected

Hirota K et al, J Exp Med , 2007

• Relevance from mouse models will need to be demonstrated specifically in humans

What are the plausible hypotheses out there?

• HLA molecules alone or together with other AS genes determine the microbiome

• HLA B27 transgenic Lewis rats

– AS features with no colitis

– Key differences in gut microbiota compared to wild type

– 16s rDNA sequencing and short fragment sequencing

Unpublished data by Lin, Asquith, Taurog et al

Advances in technology have lowered the cost of next generation sequencing

Does HLA B27 dictate an immune response to an autoantigen?

• No autoantigen identified to explain pathogenesis of AS

• B27 dimerizes on cell surface and can trigger NK cell response

• HLA B27 within cytosol is relatively unstable and can trigger series of events – Unfolded protein response

Taurog et al, J Rheumatol, 2010

Could above mechanisms cause alterations in microbiome?

If HLA molecules shape the microbiome, how could this result in disease?

• Changes in microbiome leads to change in gut permeability

• Leakage of multiple bacterial products which could be arithrogenic

HLA B27 transgenic rat shows increased intestinal permeabilityAlso demonstrated in patients with AS

Schepens et al, J Nutr 2009Martinez- Gonzalez et al, Br J Rheumatol 1994Vaile et al, J Rheumatol 1990

But which comes first?Change in permeability or change in microbiome????

SpA patients have altered microbiota and immune responsiveness to enteric organisms

Methods:25 children with ERA and 13 controlsStool and blood collected16S rDNA sequencing performedIgA and IgG ELISAs performed on select species of bacteria

Conclusion:Decreased F prauznitzii in stools of patients with ERA compared to controls(3.8% vs 10% P=0.008)Bacteroides and A muciniphilaidentified as associative agents in subsets of ERA patients

Differences in the humoral responses to these bacteria may contribute to disease

Stoll et al, Arthritis Research and Therapy, 2014

Discrete Microbial signature of terminal ileum of patients with AS compared to healthy controls

Costello et al, Arthritis and Rheumatology, 2015

Methods:Microbial profiles for terminal ileum biopsy specimens compared between AS and controlsUsing 16s ribosomal RNA gene sequencing and analysis techniques

Results:Terminal ileal microbial communities differ significantly (P<0.001) from those in healthy subjects

Driven by higher abundance of 5 families of bacteria and decrease in abundance of 2 families of bacteria

Take home messages

• Microbiome is integral to health and disease

• More work needed to confirm how HLA B27 alters the gut microbiome

• Which microbiome population is most important and its function is a mystery

• Understanding microbiome will lead to novel insights into diseases such as IBD and AS

• Is there a role for FMT in SpA?